The present invention is directed to formulations and methods for making arachidonic acid metabolite-associated liposomes. Such arachidonic acid metabolites also include their structural analogs, synthetic enzyme inhibitors, and arachidonic acid itself. One class of such arachidonic acid metabolite is the group of bioactive agents known as the prostaglandins. This invention specifically discloses prostaglandin-associated liposomes, using prostaglandin E.sub.1. The term prostaglandin also includes synthetic compounds structurally related to the naturally occurring prostaglandins.
The prostaglandins are substances found in essentially all human tissues and body fluids and produce a broad spectrum of effects embracing practically every biological function. These substances are derived from the 20-carbon essential fatty acids (arachidonic acid, the most abundant precursor) and are biologically synthesized into structures such as the 20-carbon prostaglandin subclass E.sub.1 ("PGE.sub.1 ") shown below: ##STR1## Other prostaglandin subclasses are designated by letters and distinguished by substitutions on the cyclopentane ring. Such subclasses are the prostaglandin E and F.sub.a series, which have been most intensively studied, with the subclasses A, B, and C being derivatives of the E's. Prostaglandins A through F are considered the "primary prostaglandins"; the structures of prostaglandins G.sub.2 and H.sub.2 (the cyclic endoperoxides) and thromboxanes A.sub.2 and B.sub.2 have been more recently elucidated (Goodman et al., eds., The Pharmacological Basis of Therapeutics, MacMillan Publishing Co., New York, pp. 668-676). Other bioactive agents that may be used in the invention are prostacyclines and leukotrienes.
The prostaglandins have diverse physiological actions such as vasodilative action, improvement of peripheral blood circulation, and antilipolysis. The prostaglandins are therapeutically indicated in many conditions, including but not limited to ductus arteriosus, stimulation of uterine contractions leading to induction of labor at term as well as abortion, treatment of bronchial asthma, and suppression of gastric ulceration in animals. They are also used in the prophylaxis of arteriosclerosis.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "head" orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys, Acta., 1968, 135:624-638), and large unilamellar vesicles.
Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO 86/00238, Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter. Vesicles may also be made by an extrusion technique through a 200 nm filter, such vesicles are known as VET.sub.200 s.
Another technique used to form liposomes is the "reverse phase evaporation" (REV) process of Papahadjopoulos (U.S. Pat. No. 4,235,871, issued Nov. 25, 1980). Such process forms oligolamellar lipid vesicles wherein the aqueous material to be encapsulated is added to lipids in organic solvent, forming an water-in-oil type emulsion. The organic solvent is removed, forming a gel. The gel is dispersed in aqueous medium converting it to a suspension. Yet another technique is the detergent-dialysis process (Enoch et al., 1979, Proc. Natl. Acad. Sci., 76:145). In this process, lipid is mixed with a detergent such as deoxycholate in aqueous solution, sonicated, and the detergent removed by gel filtration. A further technique is the ethanol infusion technique of Batzri et al. (1973, Biochim. Biophys. Acta., 298:1015), for forming small unilamellar vesicles, whereby an ethanol solution of lipid is injected into the desired aqueous phase, forming liposomes of about 30 nm to about 2 um in diameter. The residual ethanol may then be removed by rotoevaporation.
Another class of liposomes that may be used in the present invention are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies". The relevant portion of these references is incorporated herein by reference.
In a liposome-drug delivery system, a bioactive agent such as a drug is entrapped in or associated with the liposome and then administered to the patient to be treated. For example, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schnieder, U.S. Pat. No. 4,114,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
The use of liposomes to administer drugs has raised problems with regard to both drug encapsulation and drug release during therapy. With regard to encapsulation, there has been a continuing need to increase trapping efficiencies so as to minimize the lipid load presented to the patient. In addition, high trapping efficiencies mean that only a small amount of drug is lost during the encapsulation process, an important advantage when dealing with expensive drugs. As to drug release, many drugs have been found to be rapidly released from liposomes after encapsulation. Such rapid release diminishes the beneficial effects of liposome encapsulation. Accordingly, there have been continuing efforts by those skilled in the art to find ways to reduce the rate of release of drugs from liposomes.
In addition to these problems with encapsulation and release, there is the overriding problem of finding a commercially acceptable way of providing drug-containing liposomes to the clinician. Although the production and loading of liposomes on an "as needed" basis is an acceptable procedure in an experimental setting, it is, in general, unsatisfactory in a clinical setting. Accordingly, there is a significant and continuing need for methods whereby liposomes, with or without encapsulated drugs, can be shipped, stored and in general moved through conventional commercial distribution channels without substantial damage.